A fluorescence-based reporter for monitoring expression of mycobacterial cytochrome bd in response to antibacterials and during infection

Abstract

Cytochrome bd is a component of the oxidative phosphorylation pathway in many Gram-positive and Gram-negative bacteria. Next to its role as a terminal oxidase in the respiratory chain this enzyme plays an important role as a survival factor in the bacterial stress response. In Mycobacterium tuberculosis and related mycobacterial strains, cytochrome bd is an important component of the defense system against antibacterial drugs. In this report we describe and evaluate an mCherry-based fluorescent reporter for detection of cytochrome bd expression in Mycobacterium marinum. Cytochrome bd was induced by mycolic acid biosynthesis inhibitors such as isoniazid and most prominently by drugs targeting oxidative phosphorylation. We observed no induction by inhibitors of protein-, DNA- or RNA-synthesis. The constructed expression reporter was suitable for monitoring mycobacterial cytochrome bd expression during mouse macrophage infection and in a zebrafish embryo infection model when using Mycobacterium marinum. Interestingly, in both these infection models cytochrome bd levels were considerably higher than during in vitro culturing of M. marinum. The expression reporter described here can be a valuable tool for elucidating the role of cytochrome bd as a survival factor.

Figure 1

Construction and basic characterization of the cytochrome bd expression reporter. (A) Genetic construction of the reporter plasmid with the mCherry gene under control of the cydA promoter. (B) Growth of Mycobacterium marinum carrying the cydA reporter in 7H9 medium. Three independent biological replicates were monitored for growth. Error bars indicate the standard deviation (s.d.) value. (C) mCherry fluorescence emitted by the cydA reporter during in vitro culture under aerated (normoxia) conditions and hypoxic conditions. Three independent biological replicates were monitored. The fluorescence signal was corrected for autofluorescence of WT bacteria without a plasmid. Error bars indicate the s.d. (D) cydA induction in response to the nitric oxide donor DETA-NO (1x MIC and 10x MIC) on day 3 after induction. Three independent biological replicates were monitored. Error bars indicate the s.d. value.

Figure 2

Selected drugs are active in a zebrafish embryo infection model 3 days post treatment. (A) Infected fish, untreated (top) brightfield and fluorescence image as compared to a control, uninfected fish. (B) BDQ treated fish. (C) Treatment with Q203. (D) treatment with CFZ. Zebrafish infection experiments were performed with at least three biological replicates.

Figure 3

Differential regulation of cydA in response to antibacterials analyzed by flow cytometry. A histogram plot showing the number of events (y-axis). Peak height indicates mCherry fluorescence intensity (x-axis) for: Untreated samples (red line), 1x MIC of the shown antibiotic (blue line) and 10x MIC (green line). WT without cydA reporter served as a background control (black line). Three independent experiments were performed, each histogram represents data from one representative experiment with 30,000 cells.

Figure 4

Time- and concentration dependency of cydA induction by inhibitors of oxidative phosphorylation. (A) Response to BDQ for 1x, 10x and 100x MIC over time. (B) Response to Q203 for 1x, 10x and 15x MIC. (C) Response to CFZ for 1x, 10x and 60x MIC. The fold inductions were calculated by dividing the mean fluorescence intensities of the treated samples by the corresponding untreated controls. The data of three independent biological replicates were used. Error bars represent the s.d. values.

Figure 5

The cydA reporter in a mouse macrophage infection model. (A) The percentage of infected RAW cells over time for NT (non-treated) as compared to cells that were treated with 1x or 10x MIC BDQ. The experiment was performed in triplicate and error bars represent s.d. values. (B) Flow cytometry experiment determining the ratio of mCherry signal (MFI) relative to the mEos3.1 signal (MFI) for bacteria in culture compared to values obtained from mouse macrophage cell infection. Data represents triplicate measurements and error bars represent s.d. values. (C) Normalized mCherry MFI over time during cell infection, comparing non-treated (NT) to BDQ-treated infected macrophages. The experiment was performed in triplicate and s.d. values are indicated by the error bars.

Figure 6

The cydA reporter in a zebrafish embryo model. Confocal microscopy images of casper zebrafish embryos at maximum projection. Time course of mEos3.1 and mCherry signals during infection (dpi = days post infection). Upper row: mEos3.1 signal indicates infection. Middle row; mCherry fluorescence indicates cydA transcription. Lower row: overlay of upper and middle row. Scale bars, 100 µm. Three independent experiment were performed, the images were selected from a representative experiment.